Approximately 1.4 million Americans are diagnosed with cancer each year. In addition, approximately 550,000 Americans die from cancer each year in the United States alone. Prostate and lung cancers are the leading causes of death in men while breast and lung cancer are the leading causes of death in women.
Cellular differentiation, growth, function and death are regulated by a complex network of mechanisms at the molecular level in a multicellular organism. In the healthy animal or human, these mechanisms allow the cell to carry out its designed function and then die at a programmed rate.
Abnormal cellular proliferation, notably hyperproliferation, can occur as a result of a wide variety of factors, including genetic mutation, infection, exposure to toxins, autoimmune disorders, and benign or malignant tumor induction.
A tumor, also called a neoplasm, is a new growth of tissue in which the multiplication of cells is uncontrolled and progressive. A benign tumor is one that lacks the properties of invasion and metastasis and is usually surrounded by a fibrous capsule. A malignant tumor (i.e., cancer) is one that is capable of both invasion and metastasis. Malignant tumors also show a greater degree of anaplasia (i.e., loss of differentiation of cells and of their orientation to one another and to their axial framework) than benign tumors.
Proliferative disorders are currently treated by a variety of classes of compounds including alkylating agents, antimetabolites, natural products, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists.
Natural and synthetic small molecules that target the microtubule cytoskeleton have proven invaluable in understanding the mechanisms of cell division. Modulators of the microtubule polymerization (such as colcimid and paciltaxel) interfere with cell division by altering microtubule dynamics, resulting in extended mitotic arrest and cell death (Jordan and Wilson, 2004; Zhou and Giannakakou, 2005). Microtubule disrupters have found use as chemotherapeutics, but because microtubules are essential for many cellular functions, toxicity issues limit their use as anti-cancer agents. Kinesins may be potential targets for anti-proliferative drugs (Bergnes et al., 2005; Duhl and Renhowe, 2005). Kinesins are motor proteins associated with microtubules. There are roughly 14 different kinesins (Lawrence et al., 2004), whose functions range from vesicular transport to cell division. RNAi analysis of 25 Drosophila kinesins revealed that four members were involved in mitotic spindle assembly (Goshima and Vale, 2003). One of these is the class five kinesin spindle protein (KSP) and is referred to in the literature as Kinesin 5, KSP or Eg5. Eg5 is a plus-end directed motor separates the spindle poles early in mitosis (Blangy et al., 1995). Eg5 is antagonized by minus-end directed kinesin known as HSET or NCD (Mountain et al., 1999), and inhibition of Eg5 results in spindle collapse and formation of a monopolar spindle. Because chromosomes are unable to form normal bipolar attachments to the spindle, the cell arrests in mitosis, and eventually dies by apoptosis (Skoufias et al., 2006). Although there are reports that Eg5 plays a role in neuronal development (Haque et al., 2004), there are no other known functions outside of mitosis in adults, and disruption of Eg5 has no effect on interphase microtubule dynamics or organization (Mayer et al., 1999b; Hotha et al., 2003; DeBonis et al., 2004).
Several Eg5 inhibitors, including monastrol (Mayer et al., 1999a), S-trityl-L-cysteine (2.5) (STLC) (Mayer et al., 1999b; Hotha et al., 2003; DeBonis et al., 2004) and more recently Ispinesib (Luo et al., 2007) have been identified. All act as specific, allosteric inhibitors of Eg5 ATPase activity (Brier et al., 2004; Brier et al., 2006a; Skoufias et al., 2006), and exhibit no inhibitory activity against the other kinesin family members (Mayer et al., 1999b; Skoufias et al., 2006). All three molecules bind an allosteric site in between a helix 3 and Loop 5 domain of the Eg5 motor domains (Turner et al., 2001; Maliga et al., 2002; Brier et al., 2004; Brier et al., 2006a; Brier et al., 2006b; Maliga and Mitchison, 2006; Maliga et al., 2006), and it has been proposed that the longer Loop 5 domain found in Eg5 class 5 kinesins explains in part the specificity of these drugs to class 5 kinesins (Turner et al., 2001). And while the kinetics of inhibition are different between these molecules, all appear to act through a similar mechanism where ADP release is perturbed without blocking motor domain release from the microtubule (Cochran and Gilbert, 2005; Skoufias et al., 2006; Lad et al., 2008). All three inhibitors have demonstrated the ability to kill cancer cells (Mayer et al., 1999b; Skoufias et al., 2006; Luo et al., 2007), and while monastrol is not effective at pharmacologically relevant concentrations, Ispinesib is currently in phase II clinical trials as a treatment directed to a number of malignancies (Beer et al., 2008; Lee et al., 2008) (check reviews for additional citations).
STLC is a derivative of the amino acid cysteine, with a trityl group linked to the sulfur on the cysteine side chain. STLC has almost a hundred fold lower IC50 than monastrol in vitro and in vivo (DeBonis et al., 2004; Skoufias et al., 2006), and cells exposed to STLC undergo a reversible mitotic arrest that lasts up to 72 hours before cells undergo apoptosis (DeBonis et al., 2004; Brier et al., 2006a). Eg5 mutants that confer monastrol resistance also confer resistance to STLC, lending further support to the notion that STLC binds the same allosteric site as monastrol (Brier et al., 2006a; Maliga and Mitchison, 2006). The cell permeability of STLC is facilitated by the non-polar trityl group, since the carboxylic acid and amine groups are charged to form a zwitterionic species at physiological pH. Thus, while the non-polar trityl group greatly increases cell permeability, the charged groups are likely accounting for the relatively high IC50 for STLC. Moreover, compound 2.5 (STLC) as well as the other Eg5 inhibitors appear to be specific to vertebrate Eg5 isoforms, thus limiting its use in nonmammalian model systems.
S-Trityl-L-cysteine (STLC) is a known inhibitor of Eg5. (Zee-Cheng et al. Experimental antileukemic agents: Preparation and structure-activity study of S-tritylcysteine and related compounds. J. Med. Chem. (1970), 13 (3), 414-18; Zee-Cheng, et al. Structural modification of S-trityl-L-cysteine. Preparation of some S-(substituted trityl)-L-cysteines and dipeptides of S-trityl-L-cysteine. J. Med. Chem. (1972), 15 (1), 13-16; David Kessel, et al. Effects of S-(trityl)-L-cysteine and its analogs on cell surface properties of leukemia L1210 cells. Biochemical Pharmacology (1976), 25 (16), 1893-7; Sebastien Brier, et al. Molecular Dissection of the Inhibitor Binding Pocket of Mitotic Kinesin Eg5 Reveals Mutants that Confer Resistance to Antimitotic Agents. Journal of Molecular Biology (2006), 360 (2), 360-376. Naohisa Ogo, et al. (2007). Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorg. Med. Chem. Lett. (2007) 17, 3921-3924; Emmanuel Klein, et al. New chemical tools for investigating human mitotic kinesin Eg5. (2007) Bioorg. Med. Chem. Lett. (2007), 17, 6474-6488; and Salvatore DeBonis, et al. Structure-Activity Relationship of S-Trityl-L-Cysteine Analogues as Inhibitors of the Human Mitotic Kinesin Eg5. J. Med. Chem. (2008), 51 (5), 1115-1125. Closely related derivatives have been proposed as agents for the treatment of cell proliferative disease. (Akira Asai, et al. Eg5 inhibitor and agent for treatment of cell proliferative disease containing the same. PCT Int. Appl. (2008), 34 pp. CODEN: PIXXD2 WO 2008114505 A1 20080925 CAN 149:370564 AN 2008:1157799 CAPLUS).